ATTACHMENT I--FINAL RISK ASSESSMENT OF ESCHERICHIA COLI K-12 DERIVATIVES (February 1997) I. INTRODUCTION Escherichia coli is one of a number of microorganisms which are normal inhabitants of the colons of virtually all warm- blooded mammals. E. coli belongs to the taxonomic family known as Enterobacteriaceae, which is one of the best-defined groups of bacteria. The strain E. coli K-12 is a debilitated strain which does not normally colonize the human intestine. It has also been shown to survive poorly in the environment, has a history of safe commercial use, and is not known to have adverse effects on microorganisms or plants. Because of its wide use as a model organism in research in microbial genetics and physiology, and its use in industrial applications, E. coli K-12 is one of the most extensively studied microorganisms. History of Commercial Use and Products Subject to TSCA Jurisdiction E. coli K-12 has a history of safe use. Its derivatives are currently used in a large number of industrial applications, including the production of specialty chemicals (e.g., L- aspartic, inosinic, and adenylic acids) and human drugs such as insulin and somatostatin (Dynamac, 1990). Further, E. coli can produce a number of specialty chemicals such as enzymes which would be regulated under TSCA. An insulin-like hormone for use as a component of cell culture media, resulting from a fermentation application in which E. coli was used as the recipient, has already been reviewed under TSCA (Premanufacture Notice P87-693). EPA recently reviewed a submission (94-1558) for use of E. coli K-12 to produce indigo for use as a dye. In general, E. coli K-12 is one of the most extensively studied bacteria, and has been used in genetic studies in laboratories worldwide. Experience with the use of E. coli is reflected in its classification under the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines, U.S. Department of Health and Human Services, 1986). With the exception of strains which are known to be pathogenic, E. coli is considered a Class 1 Agent under the NIH Guidelines. Class 1 consists of all organisms which are not either human or animal pathogens. Most experiments involving E. coli K-12 have been exempted from the NIH Guidelines based on an analysis of safety, 2 except in certain circumstances (see Appendix C-II of the NIH Guidelines). Moreover NIH, under section III-D-4 of the NIH Guidelines, exempts transfers of genetic material between species that exchange DNA by known physiological processes with the genus Escherichia. Included in this exemption are exchanges between Escherichia and the closely related genera of Shigella, Salmonella, Enterobacter, Citrobacter, Klebsiella, Erwinia, Pseudomonas aeruginosa; also included are the species Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, and Yersinia enterocolitica. II. IDENTIFICATION AND TAXONOMY A. Overview E. coli belongs to the family Enterobacteriaceae. All Enterobacteriaceae are defined as Gram-negative, non-sporeforming rods that are facultative anaerobes. During the 1960's and 1970's, large amounts of information were generated regarding the phenotypic characteristics of the Enterobacteriaceae. The reasons for this increase in knowledge were two-fold. First, beginning in the early 1970's, a number of methods became available for the identification of enteric bacteria. These methods were based on biochemical or phenotypic reactions and could be performed with minimum labor and cost. Second, a major shift in nosocomial infections from Gram-positive to Gram- negative bacteria occurred in hospital patients during the 1960's and the early 1970's. Therefore, clinical microbiology laboratories, faced with the pressing need for accurate identification systems for enteric bacteria, carried out an extensive characterization of the members of this group of bacteria, including E. coli. B. Taxonomy and Characterization Escherichia coli is a member of the family Enterobacteriaceae and has been described by Brenner (1984). Escherichia is a Gram-negative rod which can be motile by peritrichous flagella or nonmotile. Escherichia is also a facultative anaerobe which has both a respiratory and a fermentative type of metabolism, and commonly occurs in the intestinal tract of humans and other animals. E. coli K-12 was originally isolated from a convalescent diphtheria patient in 1922 (Bachmann, 1972). Because it lacks virulence characteristics, grows readily on common laboratory media, and has proven to be a valuable tool for microbial physiology and genetics research, it has become the standard bacteriological strain used in microbiological research and teaching. E. coli K-12 is now considered an enfeebled organism 3 as a result of being maintained in the laboratory environment for over 70 years (Williams-Smith, 1978). E. coli can be readily differentiated from closely related bacteria by a number of standard tests. Classically, this has been accomplished by testing for production of indole from tryptophan, production of acid from glucose media using the dye methyl red as an indicator, lack of production of acetoin as a metabolic endproduct (also known as the Voges-Proskauer reaction), and the inability to utilize citrate as a sole source of carbon. Collectively, these reactions are known as the IMViC battery. The IMViC battery was developed for the analysis of water samples where it was important to differentiate E. coli, which was found to be always associated with fecal contamination of water, from other closely related bacteria which could be found naturally in water sources. Further refinements of the IMViC tests are used today and are available as commercial test kits. C. Related Species of Concern Taxonomically, the four species of the genus Shigella are closely related to E. coli. Shigella species cause diarrhea in humans and are classified as Class 2 agents under the NIH Guidelines. The Shigella species and E. coli share a high level of DNA sequence homology and many protein and polysaccharide capsular antigens. [These capsular antigens can be used to distinguish between E. coli strains and the pattern of capsular antigens determine the organism's "serotype" (Smith 1977).] The two genera can be distinguished based on the fact that E. coli has a unique colony morphology when grown on certain differential laboratory media (Jawetz et al., 1987). Commercially prepared kits for distinguishing between these organisms are available. Most E. coli serotypes are benign and may even contribute to normal function and nutrition in the gastrointestinal tract. A few E. coli serotypes are pathogens. E. coli K-12 strains in use today are from standard culture collections (Bachmann, 1972), such as the American Type Culture Collection and are not recent environmental isolates. As a result, these K-12 strains are well-characterized and should be expected to remain as pure cultures under standard microbiological practices. K-12 strains are distinguishable from both Shigella and other Escherichia (Cooke, 1974, Orskov 1978, Schmidt 1973). III. HAZARD ASSESSMENT The Proceedings of the Falmouth Workshop held in June 1977 served as a primary source for this assessment (Gorbach, 1978). 4 A. Human Health Hazards The potential of K-12 strains to present risks to human health are examined in this assessment by analyzing K-12 in terms of (1) the phenotypic traits relevant to colonization of the colon, and (2) toxin production. 1. Colonization and Pathogenicity E. coli is an inhabitant of the human colon, and it is thought that the primary means through which humans acquire their intestinal flora is through ingestion. Workers in fermentation facilities would not be expected to ingest E. coli under standard good practice, which prohibits the ingestion of food in work areas; however, some inhaled bacteria could be swallowed. In order to evaluate K-12's potential to colonize the human intestine the following should be addressed: (1) the characteristics relevant to E. coli colonization of the human colon, and how K-12 compares to other E. coli in terms of these traits, and (2) data relevant to colonization potential of K-12 strains. The binding of an E. coli to the mucosal surface of the colon requires two factors. The first factor is the production of a specific glycocalyx or fimbriae from the surface of the bacterium. This specific glycocalyx recognizes a specific lectin on the surface of the enterocyte lining of the human colon. The glycocalyxes appear to bind to structures such as the mucus glycoproteins elaborated from the goblet cells of the intestine. In Gram-negative bacteria, the polysaccharide chains arising from the core of the lipopolysaccharides in the outer membrane appear to be the major ones which affect binding to the colon. E. coli K-12 is defective in at least three cell wall characteristics. The outer membrane has a defective lipopolysaccharide core which affects the attachment of the O- antigen polysaccharide side chains (Curtiss, 1978). Second, it does not have the type of glycocalyx required for attachment to the mucosal surface of the human colon (Edberg, 1991) as a result of the altered O-antigen properties noted above. Finally, K-12 strains do not appear to express capsular (K) antigens, which are heat-labile polysaccharides important for colonization and virulence (Curtiss, 1978). K-12, thus, is not able to colonize the human intestinal tract under normal conditions, even after ingestion of billions of organisms (Anderson, 1975, Cohen et al., 1979., Levy and Marshall, 1981; Levy et. al, 1980, Smith, 1975). As noted above, K-12 is defective in cell wall components relevant to the ability 5 to recognize and adhere to the mucosal surface of colonic cells (Curtiss, 1978). The normal flora in residence in the colon thus can easily exclude K-12, and prevent it from colonizing the human colon. A number of experiments have been conducted to measure the ability of K-12 to colonize in comparison to other E.